PROJECT SUMMARY Despite the important role of the tumor stroma in cancer progression, the vast majority of anticancer therapies target cancer cells and not the tumor microenvironment. A novel drug delivery approach that not only target tumor cells but also the inflammatory cells would be highly desirable. Here we propose to develop a delivery platform that builds on our recent work designing single chain variable fragment antibodies that both inhibit inflammatory monocyte migration to tumors and polarize macrophages towards the tumoricidal M1 phenotype by hijacking the CCL2/CCR2 pathway. This approach capitalizes on antibody binding specificity but exploits CCR2 receptor structure and function to improve therapeutic efficacy. We hypothesize that simultaneous targeting of multiple CCR2 epitopes will mediate a synergistic drug effect and inhibit tumor growth and metastasis by inducing M1 macrophage polarization and reducing macrophage migration. Our protein delivery approach capitalizes on both the targeting specificity of antibody fragments and also their ability to induce or modify downstream signaling. Thus, the antibody fragments will serve both as a delivery vehicle and therapeutic drug and have the potential to minimize carrier-induced toxicity. The specific aims of this proposal are to (1) synthesize a modular CCR2-targeting zip-ligand system for macrophage polarization and establish its mechanism of action; (2) assess the effects of multi-epitope CCR2-targeting constructs on macrophage polarization, tumor growth, and metastasis in a triple-negative breast cancer (TNBC) mouse model; and (3) evaluate the therapeutic effects of multi-epitope CCR2 targeting in combination with (a) anti-PD-1 blockade and (b) other therapeutics in a mouse model of TNBC, a subtype with particularly poor clinical outcomes and limited treatment options. Upon successful completion, this project will establish a new delivery concept to modify downstream signaling and modulate cellular phenotypes.